Characterizing interactions of proteins is pivotal for understanding their function. Recently, single-molecule imaging-based methods have proven useful for directly testing the stoichiometry of multi-subunit protein complexes. A limiting factor is the labeling of proteins with multiple spectrally discernible tags and low background. Here, we describe the use of zinc-finger (ZF)-mediated protein labeling for single-molecule imaging studies in living cells. A DNA-binding ZF is fused to the protein of interest and labeled by a DNA probe carrying the specific ZF binding sequence and an organic dye. Nonspecific binding is minimized by injecting the DNA/dye conjugate into the cell. With a reproducible labeling efficiency of 20%, we developed an approach to deduce the multiplicity of the subunits in a protein complex from the overall degree of labeling. We were able to confirm the fixed 2:2 assembly of the NMDA receptor in a three-color single-molecule imaging setup and reject alternative stoichiometries. Due to the modular design and small size of ZF proteins, this approach will allow the analysis of more complicated protein interaction patterns to understand the assembly rules for large protein complexes.

中文翻译：

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锌指介导的标记揭示了膜蛋白的化学计量。

表征蛋白质相互作用对于理解其功能至关重要。最近，已证明基于单分子成像的方法可用于直接测试多亚基蛋白质复合物的化学计量。一个限制因素是具有多个光谱可识别标签且背景低的蛋白质标记。在这里，我们描述了锌指（ZF）介导的蛋白质标记在活细胞中单分子成像研究中的使用。将结合DNA的ZF与目标蛋白质融合，并通过带有特定ZF结合序列和有机染料的DNA探针进行标记。通过将DNA /染料偶联物注射到细胞中，可以将非特异性结合减至最少。我们以20％的可复制标记效率，开发了一种从整体标记程度推论蛋白质复合物中亚基多样性的方法。我们能够在三色单分子成像设置中确认NMDA受体的固定2：2组装，并拒绝其他化学计量。由于ZF蛋白的模块化设计和小尺寸，这种方法将允许分析更复杂的蛋白相互作用模式，以了解大蛋白复合物的组装规则。